CN109943515A - It is a kind of produce carboxy-lesterase recombinant bacterium and its application - Google Patents
It is a kind of produce carboxy-lesterase recombinant bacterium and its application Download PDFInfo
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- CN109943515A CN109943515A CN201910360379.XA CN201910360379A CN109943515A CN 109943515 A CN109943515 A CN 109943515A CN 201910360379 A CN201910360379 A CN 201910360379A CN 109943515 A CN109943515 A CN 109943515A
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Abstract
The invention discloses a kind of recombinant bacterium for producing carboxy-lesterase and its applications, belong to enzyme engineering field.The present invention is respectively wherein 13831.8U/L and 8135.9U/L using 1- naphthyl acetate and 2- naphthyl acetate as the enzyme activity of substrate in crude enzyme liquid by amino acid sequence carboxy-lesterase BaCEs02 heterogenous expression in Escherichia coli as shown in SEQ ID NO.1.It purifies the obtained pure enzyme solution of carboxy-lesterase BaCEs02 and 92.2%, 95.6%, 87.3% is up to respectively to the degradation rate of phthalate (diethyl phthalate, dibutyl phthalate or diisobutyl phthalate), provide a kind of method of efficient degradation plasticiser phthalate.
Description
Technical field
The present invention relates to a kind of recombinant bacterium for producing carboxy-lesterase and its applications, belong to enzyme engineering field.
Background technique
Carboxy-lesterase (carboxylesterase, EC 3.1.1.1) be refer to catalyzing hydrolysis carboxylate generate carboxylic acid and
The nonspecific esterase of alcohol.Carboxy-lesterase is widely used in production and real life, and is had been obtained more and more existing
For the favor of the relevant industries such as pharmaceutical industries, chipal compounds synthesis and fine chemistry industry.Using carboxy-lesterase as catalyst
It is catalyzed chiral molecules synthesis, such as production naproxen and 2- aryl naphthoic acid；Carboxy-lesterase is also dropped as green catalyst simultaneously
Solve the pesticide residue in soil and the plasticiser phthalate in soil, water body.Due to originating from the carboxylate of animals and plants
Enzyme there is enzyme activity it is low, yield is few, extraction is complicated, thermal stability is poor the defects of, and microbe-derived carboxy-lesterase have it is good
Good regioselective and stereoselectivity, the hydrolysis of catalysis esters and amides compound that can be efficiently mild, Lipase absobed
It reacts with transesterification etc., all has in related fieldss such as food industry, bio-decontaminated agent, medicine, energy development and environmental protections
Wide application prospect.
Carboxy-lesterase is present in the actinomyces of fungi, bacterium and individual species in microorganism.Fungi carries wherein
Important function, 12 belong to 23 kinds and can produce carboxy-lesterase in fungi；Due to the carboxy-lesterase expression quantity of originated from fungus is few, enzyme activity compared with
It is low, and the carboxy-lesterase of bacterial origin is easy to get, zymologic property is excellent, high catalytic efficiency, so the carboxylate of bacterial origin
Enzyme has been to be concerned by more and more people.Carboxy-lesterase is primarily present in including streptococcus, lactobacillus, pseudomonad in bacterium
In.And the expression quantity of carboxy-lesterase is lower in wild mushroom, is not able to satisfy industrial application；As genetic engineering is applied to carboxy-lesterase
Research in, many carboxylesterase genes have obtained heterogenous expression.2000, Kademi, A. et al. was in Bacillus circulans
Cloning enzyme activity is 88.2Umg-1Moderate is thermophilic carboxy-lesterase, 2001, N ú ria Prim et al. was in bacillus subtilis
Cloning enzyme activity is 2.28Umg-1Type B carboxy-lesterase.2003, K.M.Fenster et al. cloned enzyme activity in lactic acid bacteria
For 564Umg-1Acidic carboxy-late's enzyme.But these carboxy-lesterase enzyme activity times are so unable to reach industrial demand.Therefore,
The carboxy-lesterase for obtaining the bacterial origin of high enzyme activity will have highly important value and significance in industrial application.
Summary of the invention
The present invention is different in Escherichia coli by amino acid sequence carboxy-lesterase (BaCEs02) as shown in SEQ ID NO.1
Source expression using 1- naphthyl acetate and 2- naphthyl acetate as the enzyme activity of substrate is respectively 13831.8U/L and 8135.9U/ in crude enzyme liquid
L, the carboxy-lesterase purified (BaCEs02) are up to respectively using 1- naphthyl acetate and 2- naphthyl acetate as the specific enzyme activity of substrate
1680.58U·mg-1And 939.42Umg-1。
The first purpose of the invention is to provide a kind of recombinant bacteriums for producing carboxy-lesterase, are host with Escherichia coli, expression
Amino acid sequence carboxylesterase gene as shown in SEQ ID NO.1.
In one embodiment of the invention, the nucleotide sequence of the carboxylesterase gene such as SEQ ID NO.2 institute
Show.
It in one embodiment of the invention, be using pColdII is expression vector in expression in escherichia coli.
A second object of the present invention is to provide a kind of method for producing carboxy-lesterase, the method is using the recombinant bacterium
Carry out fermenting and producing.
Third object of the present invention is to provide a kind of methods of phthalate of degrading, and are complete with the recombinant bacterium
Cell or the carboxy-lesterase of its production are catalyst, phthalate of degrading.
In one embodiment of the invention, the phthalate are as follows: diethyl phthalate, adjacent benzene two
Formic acid dibutyl ester or diisobutyl phthalate.
In one embodiment of the invention, the degradation condition of the carboxy-lesterase are as follows: pH5.0~8.0.
In one embodiment of the invention, the degradation condition of the carboxy-lesterase is preferred are as follows: pH 6.5.
In one embodiment of the invention, the degradation temperature of the carboxy-lesterase are as follows: 15~60 DEG C.
In one embodiment of the invention, the degradation temperature of the carboxy-lesterase is preferred are as follows: 45 DEG C.
In one embodiment of the invention, the degradation time is 1~6h.
In one embodiment of the invention, the degradation specifically: by the recombinant bacterium or the carboxylate of its production
Enzyme enzyme solution is added to phthalate (diethyl phthalate, dibutyl phthalate of final concentration of 1~10mM
Or diisobutyl phthalate) in, in 15~60 DEG C of 1~10h of water-bath.
In one embodiment of the invention, the degradation is more specifically: the carboxy-lesterase enzyme solution is added to end
Concentration is phthalate (two isobutyl of diethyl phthalate, dibutyl phthalate or phthalic acid of 1mM
Ester) in, in disodium hydrogen phosphate-potassium phosphate buffer (pH 6.5) of 1.5mL, 1~10h is reacted at 40 DEG C.
The present invention also provides the recombinant bacterium or its production carboxy-lesterase field of environment protection application.
In one embodiment, the application is that the carboxy-lesterase is added to containing plasticiser phthalic acid ester
In the sewage of class, carboxy-lesterase degradation phthalate is utilized.
Beneficial effects of the present invention:
(1) present invention is by amino acid sequence as carboxy-lesterase BaCEs02 shown in SEQ ID NO.1 is different in Escherichia coli
Source expression, wherein in crude enzyme liquid using 1- naphthyl acetate and 2- naphthyl acetate as the enzyme activity of substrate be respectively 13831.8U/L and
8135.9U/L, the carboxy-lesterase purified (BaCEs02) using 1- naphthyl acetate and 2- naphthyl acetate as the specific enzyme activity of substrate,
It is up to 1680.58Umg respectively-1And 939.42Umg-1。
(2) the obtained pure enzyme solution of carboxy-lesterase BaCEs02 of purifying to phthalate (diethyl phthalate,
Dibutyl phthalate, diisobutyl phthalate) degradation rate respectively be up to 92.2%, 95.6%, 87.3%, provide
A kind of method of efficient degradation plasticiser phthalate.
Detailed description of the invention
Fig. 1: optimal reactive temperature.
Fig. 2: optimal reaction pH.
Fig. 3: temperature stability.
Fig. 4: pH stability.
Fig. 5: 1- naphthyl acetate enzymatic polymerization kinetics curves.
Fig. 6: 2- naphthyl acetate enzymatic polymerization kinetics curves.
Specific embodiment
(1) method that carboxy-lesterase enzyme activity is measured as substrate using 1- naphthyl acetate or 2- naphthyl acetate:
1% solid indigo plant B salt: the solid indigo plant B salt for weighing 1g, which is dissolved in distilled water, is settled to 100mL, is kept in dark place.
5% SDS: the SDS for weighing 5g is dissolved in distilled water, in 37 DEG C water-bath 1 hour, be settled to after it is completely dissolved
100mL is saved in refrigerator.
The 1- naphthyl acetate or 2- naphthyl acetate of 0.6M: the 1- naphthyl acetate or 2- naphthyl acetate for weighing 11.17g are dissolved in
In 95% ethyl alcohol, it is settled to 100mL, is kept in dark place.
Disodium hydrogen phosphate-potassium phosphate buffer: 1/15M disodium hydrogen phosphate and 1/15M potassium dihydrogen phosphate mixed configuration
To pH7.0.
The substrate 1- naphthyl acetate of 15 μ L or 2- naphthyl acetate are added to 1.5mL disodium hydrogen phosphate-potassium dihydrogen phosphate to delay
(pH 7.0) keeps the temperature 5min in 37 DEG C of water-baths in fliud flushing, and the enzyme solution of 250 μ L after purification is added, reacts 5min, and 0.5mL is added and terminates
Developing solution DBLS (1% solid indigo plant B salt is mixed with 5%SDS with 2:5), shakes up, stands 10min, measure light absorption value under 595/555nm.
Enzyme activity definition: under optimum reaction conditions, in 1min from the 1- naphthyl acetate or 2- naphthyl acetate solution of 0.6M
Enzyme amount needed for the 1- naphthols or beta naphthal of 1 μM of release is an enzyme-activity unit.
(2) measurement of carboxy-lesterase protein concentration: according to Bradford protein quantification kit method, by certain times of dilution
Several enzyme solutions is mixed with G250 dyeing liquor, with the light absorption value at microplate reader measurement 595nm, calculates albumen according to protein concentration mark song
Concentration.Specific enzyme activity (Umg-1)=enzyme activity (UmL-1) × [protein concentration (mgmL-1)]-1。
(3) phthalate high performance liquid chromatography detection condition are as follows: C18 column (4.6 × 250mm of Agilent), wave
Long 254nm, mobile phase ratio are methanol: water=80:20, and detection temperature is 30 DEG C.
(4) phthalate degradation rate calculation formula: degradation rate=residue concentration of substrate/initial substrate concentration
Embodiment 1: the building of engineered strain
The carboxylic of artificial synthesized nucleotide sequence (amino acid sequence is as shown in SEQ ID NO.1) as shown in SEQ ID NO.2
Acid esters enzyme BaCEs02 gene order.By BaCEs02 gene order and plasmid vector pColdII with I He of restriction enzyme Sac
Connection conversion obtains recombinant bacterium E.coli BL21- into E.coli BL21 (DE3) competent cell after I double digestion of Xba
pColdII-BaCEs02。
Embodiment 2: the expression and purification of carboxy-lesterase (BaCEs02)
LB culture medium g/L: sodium chloride 10, tryptone 10, Yeast Extract 5, pH 7.
Recombination bacillus coli E.coli BL21-pColdII-BaCEs02 is inoculated in containing 100mgmL-1The LB of ammonia benzyl
In fluid nutrient medium, (it is transferred in E.coli BL21 (DE3) with original strain E.coli BL21 (DE3) and unloaded bacterial strain
PClodII plasmid) as control, 37 DEG C, 200rmp cultivates 12h, and then the 500 above-mentioned seed liquors of μ L are inoculated in containing 50 μ L ammonia
In the 50mL LB culture solution of benzyl, 37 DEG C of culture 2.5h, until OD600It is 0.6, shaking table is cooled to 15 DEG C, stands 30min.Every bottle
The IPTG of the 40 final concentration of 0.4mol/L of μ L is added as inducer, inducer is not added as a control group, in 15 DEG C of 200rmp
Culture is for 24 hours.
Bacterium solution is collected, 4 DEG C, 8000rmp obtains thallus after being centrifuged 10min, and 5mL phosphate buffer (0.02mol/ is added
L, pH7.0) thallus is resuspended, Ultrasonic Cell Disruptor is broken, and supernatant is collected by centrifugation and obtains crude enzyme liquid.Crude enzyme liquid obtained above is adopted
Ni-sepharose purification is carried out with 150 protein purification system of AKTA avant and obtains BaCEs02 enzyme solution, and the concentration for measuring BaCEs02 is
29.7mg/mL is used for subsequent experimental.
Embodiment 3: the enzyme activity determination of carboxy-lesterase (BaCEs02)
In disodium hydrogen phosphate-potassium phosphate buffer (pH 7), using 2- naphthyl acetate as substrate, 15~60 DEG C of ranges
It is interior, every 5 DEG C, measure carboxy-lesterase BaCEs02 enzyme activity, it is known that the optimum temperature of carboxy-lesterase BaCEs02 is 45 DEG C (see figure
1).Under the conditions of 45 DEG C of optimal reactive temperature, in 5.0~8.0 range of pH, every 0.5, enzyme activity is measured, determines optimal reaction pH
For 6.5 (see Fig. 2).
Under optimum reaction conditions, i.e., in disodium hydrogen phosphate-potassium phosphate buffer (pH 6.5), at 45 DEG C, respectively
It as the enzyme activity of crude enzyme liquid obtained in substrate measurement embodiment 2 and is purified using 0.6M 1- naphthyl acetate and 2- naphthyl acetate
The specific enzyme activity of the carboxy-lesterase BaCEs02 arrived, wherein in crude enzyme liquid the enzyme activity of BaCEs02 be respectively 13831.8U/L and
The specific enzyme activity of 8135.9U/L, the carboxy-lesterase BaCEs02 purified respectively reach 1680.58Umg-1And 939.42U
mg-1。
Temperature stability: by 250 μ L of carboxy-lesterase (BaCEs02) enzyme solution, when pH is 6.5 respectively 10,20,30,40,
Remaining enzyme activity is measured after saving 1h in 50,60 DEG C, is set as 100% so that enzyme activity is highest.As the result is shown: carboxy-lesterase
Enzymatic activity is still maintained at 60% or more after BaCEs02 is placed one hour at 50 DEG C, has good temperature stability (see figure
3)。
1 temperature stability of table
PH stability: by 250 μ L carboxy-lesterase (BaCEs02) enzyme solutions under the conditions of temperature 45 C, respectively in pH5.0,
Remaining enzyme activity is measured after saving 1h in 5.5,6.0,6.5,7.0,7.5,8.0, is set as 100% so that enzyme activity is highest.As a result it shows
Show: enzymatic activity is maintained at 45% or more after carboxy-lesterase BaCEs02 is placed 1 hour in the range of pH5.5~7.5, has good
PH stability.
2 pH stability of table
Embodiment 4: influence of the metal ion to carboxy-lesterase (BaCEs02)
In disodium hydrogen phosphate-potassium phosphate buffer (pH 6.5) of 1.5mL, at 45 DEG C, 250 μ L are added
BaCEs02 enzyme solution, the 0.6M 2- naphthyl acetate of 15 μ L, 1mM different metal ions (Na+,K+,Zn2+,NH4 +,Mg2+,Ca2+,Cu2 +,Fe3+), measure influence of the metal ion to carboxy-lesterase BaCEs02 enzyme activity.The results are shown in Table 3, it is known that K+, NH4 +, Mg2+It is right
Enzyme activity has facilitation slightly, remaining metal ion has different degrees of inhibiting effect to enzyme.
Influence of 3 different metal ions of table to carboxy-lesterase enzyme activity
Embodiment 5: the substrate specificity of carboxy-lesterase (BaCEs02)
In disodium hydrogen phosphate-potassium phosphate buffer (pH 6.5) of 1.5mL, at 45 DEG C, 250 μ L are added
BaCEs02 enzyme solution, measurement carboxy-lesterase BaCEs02 are catalyzed 0.2-3.4mmolL-11- naphthyl acetate and 0.1-3.2mmol
L-12- naphthyl acetate reaction rate, using Origin software carry out nonlinear fitting curve Vmax and Km value is calculated,
And then Kcat/Km value is calculated.As shown in table 4, substrate 1- naphthyl acetate is compared with 2- naphthyl acetate, and carboxy-lesterase is to 1- second
Bigger (the K of the affinity of sour naphthalene estermLower, affinity is bigger), to the catalytic efficiency (K of 1- naphthyl acetatecat/Km) more preferably, reach
13.51。
The different substrate kinetic parameters of table 4
Embodiment 6: the application of carboxy-lesterase (BaCEs02)
Carboxy-lesterase (BaCEs02) enzyme solution in 50 μ L embodiments 2 after purification is taken to be added to the adjacent benzene two of final concentration of 1mM
In formate ester (diethyl phthalate, dibutyl phthalate or diisobutyl phthalate), in the phosphorus of 1.5mL
In sour disodium hydrogen-potassium phosphate buffer (pH 6.5), carboxy-lesterase (BaCEs02) enzyme solution is not added as a control group, in
After 40 DEG C of water-bath 1h, 100 μ L concentration are added in the reaction system and are that 1M HCl solution terminates reaction, then with isometric acetic acid second
Ester extraction, three parallel laboratory tests of every group of experimental setup.The amount of remaining substrate is measured, by high performance liquid chromatography to judge its hydrolysis
Degree.Carboxy-lesterase BaCEs02 is calculated to 3 kinds of plasticiser phthalates (diethyl phthalate, adjacent benzene two
Formic acid dibutyl ester, diisobutyl phthalate) degradation rate be respectively 81.2%, 88.3%, 75.7%.Know therefrom, it should
Carboxy-lesterase reaches 75% or more to the degradation rate of three kinds of plasticisers of low concentration, there is very big application valence in environment remediation
Value.
Degradation of 5 BaCEs02 of table to low concentration phthalate
Embodiment 7: the application of carboxy-lesterase (BaCEs02)
It is identical as process in embodiment 6, except that in system phthalate final concentration of 10mM.
Carboxy-lesterase (BaCEs02) enzyme solution in 50 μ L embodiments 2 after purification is added to the adjacent benzene of final concentration of 10mM
In diformic ester (diethyl phthalate, dibutyl phthalate or diisobutyl phthalate), 1.5mL's
In disodium hydrogen phosphate-potassium phosphate buffer (pH 6.5), carboxy-lesterase (BaCEs02) enzyme solution is not added as a control group,
After 40 DEG C of water-bath 1h, 100 μ L concentration are added in the reaction system and are that 1M HCl solution terminates reaction, then with isometric acetic acid
Ethyl ester extraction, three parallel laboratory tests of every group of experimental setup.The amount of remaining substrate is measured, by high performance liquid chromatography to judge its water
The carboxy-lesterase is calculated to 3 kinds of plasticiser phthalates (diethyl phthalates, O-phthalic in solution degree
Dibutyl phthalate, diisobutyl phthalate) degradation rate be respectively 55.5%, 60.4%, 43.1%.Thus result it is found that
In the case where not increasing enzyme amount, which has all reached 40% or more to the degradation rate of 3 kinds of plasticisers of high concentration.
Degradation of 6 BaCEs02 of table to high concentration phthalate
Embodiment 8: the application of carboxy-lesterase (BaCEs02)
Identical as process in embodiment 6, different hydrolysis times extend to 6h.
By the enzyme solution of 50 μ L after purification be added to final concentration of 1mM phthalate (diethyl phthalate,
Dibutyl phthalate or diisobutyl phthalate) in, enzyme solution is not added as a control group, in the phosphoric acid hydrogen of 1.5mL
In disodium-potassium phosphate buffer (pH 6.5), after 40 DEG C of water-bath 6h, 100 μ L concentration are added as the termination of 1M HCl solution
Reaction, then extracted with isometric ethyl acetate, three parallel laboratory tests of every group of experimental setup.It is surplus by high performance liquid chromatography measurement
The amount of remaining substrate, to judge its hydrolysis degree, the carboxy-lesterase is calculated is respectively to the degradation rate of 3 kinds of plasticisers
87.4%, 92.4%, 80.6%.The result illustrates the extension reaction time, and the carboxy-lesterase is to 3 kinds of plasticiser phthalic acid esters
The degradation rate of class (diethyl phthalate, dibutyl phthalate, diisobutyl phthalate) has reached 80%
More than, illustrate that extending the reaction time can be improved degradation rate.
Degradation of 7 BaCEs02 of table to low concentration phthalate
Embodiment 9: the application of carboxy-lesterase (BaCEs02)
It is identical as process in embodiment 6, except that it is 100 μ that the additive amount of carboxy-lesterase (BaCEs02) enzyme solution, which increases,
L。
The enzyme solution of 100 μ L after purification is added to phthalate (the phthalic acid diethyl of final concentration of 1mM
Ester, dibutyl phthalate, diisobutyl phthalate) in, enzyme solution is not added as a control group, in the phosphoric acid of 1.5mL
In disodium hydrogen-potassium phosphate buffer (pH 6.5), after 40 DEG C of water-bath 1h, it is that 1M HCl solution is whole that 100 μ L concentration, which are added,
It only reacts, then is extracted with isometric ethyl acetate, three parallel laboratory tests of every group of experimental setup.It is measured by high performance liquid chromatography
The amount of remaining substrate, to judge its hydrolysis degree.The amount for analyzing remaining substrate is measured through high performance liquid chromatography, and the carboxylic is calculated
Acid esters enzyme is respectively 92.2%, 95.6%, 87.3% to the degradation rate of 3 kinds of plasticisers.The result illustrates that increasing enzyme amount increases drop
Solution rate.
Degradation of 8 BaCEs02 of table to low concentration phthalate
Embodiment 10: recombinant bacterium E.coli BL21-pColdII-BaCEs02 whole-cell catalytic reaction
Disodium hydrogen phosphate-is used after recombinant bacterium E.coli BL21-pColdII-BaCEs02 obtained in embodiment 1 is collected
Potassium phosphate buffer (pH 6.5) resuspension is diluted to OD600It is 1.0, is separately added into 100 μ L bacterium solutions to final concentration 1mM neighbour's benzene
In diformic ester (diethyl phthalate, dibutyl phthalate or diisobutyl phthalate), in 40 DEG C of water
After bathing 1h, it is that 1M HCl solution terminates reaction, then is extracted with isometric ethyl acetate that 100 μ L concentration, which are added, and every group of experiment is set
Set three parallel laboratory tests.Recombination is calculated to judge its hydrolysis degree by the amount that high performance liquid chromatography measures remaining substrate
Bacterium E.coli BL21-pColdII-BaCEs02 is respectively 28.8%, 42.1% and 31.7% to the degradation rate of 3 kinds of plasticisers.
Degradation of 9 BaCEs02 of table to low concentration phthalate
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of recombinant bacterium for producing carboxy-lesterase and its application
<160> 2
<170> PatentIn version 3.3
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Claims (10)
1. a kind of recombinant bacterium for producing carboxy-lesterase, which is characterized in that with Escherichia coli be host, express amino acid sequence such as SEQ
Carboxylesterase gene shown in ID NO.1.
2. recombinant bacterium according to claim 1, which is characterized in that the nucleotide sequence of the carboxylesterase gene such as SEQ
Shown in ID NO.2.
3. recombinant bacterium according to claim 1, which is characterized in that be using pColdII be expression vector in Escherichia coli
Expression.
4. a kind of method for producing carboxy-lesterase, which is characterized in that the method is using any weight of claims 1 to 3
Group bacterium carries out fermenting and producing.
5. a kind of method for phthalate of degrading, which is characterized in that be with any recombinant bacterium of claims 1 to 3
Full cell or the carboxy-lesterase of its production are catalyst, phthalate of degrading.
6. according to the method described in claim 5, it is characterized in that, the phthalate are as follows: phthalic acid diethyl
Ester, dibutyl phthalate or diisobutyl phthalate.
7. according to the method described in claim 5, it is characterized in that, the degradation condition of the carboxy-lesterase are as follows: pH5.0~8.0.
8. according to the method described in claim 5, it is characterized in that, the degradation temperature of the carboxy-lesterase are as follows: 15~60 DEG C.
9. according to the method described in claim 5, it is characterized in that, the degradation time is 1~6h.
10. carboxy-lesterase of recombinant bacterium or its production described in claims 1 to 3 is any is in the application of field of environment protection.
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